Abstract 387: HDL-apolipoprotein A-I Exchange Occurs in the Absence of HDL Particle Remodeling

Abstract

Objective: Apolipoprotein A-I (apoA-I) spontaneously exchanges between high-density lipoprotein (HDL)-bound and lipid-free states. This is a key event in reverse cholesterol transport, necessary for de novo HDL biogenesis. However, the mechanism of HDL-apoA-I exchange (HAE) is poorly understood. In this study, we test whether HDL remodeling is obligatory to HAE or an independent process. Method and Results: Recombinant human apoA-I modified with single cysteine mutations were expressed in bacteria and labeled with Alexa 488 and Alexa 647 fluorophores. ApoA-I Alexa647 was used to synthesize rHDL particles of defined sizes with phosphatidyl choline (POPC) and cholesterol. Reconstituted HDL of 17.0, 12.2, 9.6, 8.4 and 7.8 nm were obtained. These particles were incubated with excess lipid-free apoA-I Alexa488 to initiate HAE, which was quantified using non-denaturing gradient gel electrophoresis to separate HDL particles from lipid-free apoA-I. Gels were imaged using FITC and Cy5 filters. The extent of apoA-I Alexa488 binding and apoA-I Alexa647 released from HDL was determined by densitometry. HAE occurred without significant changes in rHDL particle size for all subclasses of rHDL. HAE in the forward (lipid-free to bound) direction did not exhibit size dependence, but HAE in the reverse (lipid-bound to free) direction appeared to be size-dependent, with the 8.4 nm rHDL displaying 2-fold higher apoA-I Alexa647 dissociation compared to the 17.0 nm particle. Conclusions: The bidirectional rate of HAE can be measured simultaneously using fluorescent apoA-I probes. Reconstituted HDL particles predominantly retained their size, indicating that apoA-I exchange occurs without remodeling of HDL particles. This result has significant implications as to the possible molecular processes driving apoA-I exchange with HDL.